The ribosomal RNA in the Escherichia coli ribosome will be investigated by physical techniques to determine its three dimensional structure and to determine the nature and extent of structural change during its function. These experiments will be based on the three dimensional model of the 16S rRNA in the 30S subunit that was derived in the current grant period. Several strategies will be used to obtain structural information and monitor the internal structure; an emphasis will be on the use of photochemical reagents that can be placed at specific rRNA sites because these provide significant amounts of information about selected areas. The initial experiments will focus on the structure of the 16S rRNA around sequence positions 560, 700, 790, 900, and 1500 as these are located very close to the mRNA decoding region. This region is critical to the function of the subunit and the results obtained will distinguish between alternative rRNA foldings. When this region is solved, more peripheral regions will be investigated. The specific aims of the work are: 1. to determine the three dimensional structure of the 16S rRNA underlying the mRNA track, including three regions: at the tRNA decoding sites, at exit side of the decoding region (part of the 16S rRNA domain II) and on the entrance side of the decoding region (the 530 stem loop region of 16S rRNA), 2. to determine the effects of antibiotic binding and 16S rRNA mutations on the 16S rRNA higher order structure, 3. to determine conformational changes in the 16S rRNA higher order structure during tRNA binding, in the initiation complex with initiation factor IF3 and in the translocation process, 4. to determine if mRNA is folded as part of its path in the 30S subunit and the influence of tRNA binding and translocation on this possible folding. The ribosome is an important subject of investigation because it performs a universal biochemical process for which we do not yet know the molecular details. Increased knowledge of the ribosome will add to our understanding of the catalytic capacities of RNA, and to our general knowledge of RNA structure and ribonucleoprotein structure. An important aspect of the proposed studies is that many antibiotics exert their activity on the ribosome. It is necessary to understand the nature of these interactions and their affect on the ribosome if there is to be rational design of synthetic antibiotics.